Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has recently been appreciated that thrombin induces the retraction of endothelial cells resulting in an alteration of the integrity of the monolayers. We studied thrombin-induced changes in cytosolic calcium concentration (Ca2+i) using microfluorometry of fura-2-loaded single cells, cell topography (scanning electron microscopy), and cytoskeleton (rhodamine phalloidin) in endothelial cells. Thrombin caused an initial and sustained phase of an increase in Ca2+i. Pretreatment with pertussis toxin abolished both phases of Ca2+i response. Sustained phase of thrombin effect required extracellular calcium. Pretreatment of endothelial cells with indomethacin protracted the sustained phase, whereas a lipoxygenase inhibitor, nordihydroguaiaretic acid curtailed it. Thrombin caused a marked retraction of confluent endothelial cells coincident with the sustained phase of Ca2+i response. This was paralleled by the formation of gaps in F-actin distribution at the periphery of the cells. Pretreatment of endothelial cells with nordihydroguaiaretic acid blunted the thrombin-induced cell retraction. Microinjection of various putative messengers into the endothelial cells showed that initial Ca2+ mobilization is not sufficient to account for sustained elevation of Ca2+i. The sustained response required microinjection of phospholipase A2 or co-injection of phospholipase A2 with phosphatidylinositol 4,5-bisphosphate-specific phospholipase C, phosphatidylinositol 1,4,5-trisphosphate, or CaCl2, further implying that thrombin receptor(s) can be coupled to both phospholipases C and A2. Sustained elevation of Ca2+i was a necessary prerequisite for the thrombin-induced changes in endothelial cell topography.
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PMID:Nature of thrombin-induced sustained increase in cytosolic calcium concentration in cultured endothelial cells. 277 5

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.
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PMID:Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation. 282 9

The thrombin-dependent losses of eicosapentaenoate (EPA) from the various phospholipids of platelets derived from human subjects ingesting a fish lipid concentrate (MaxEPA) were quantitatively assessed and studied in relation to arachidonate (AA). The net loss of AA and EPA from the total phospholipid, phosphatidylcholine (PC) + phosphatidylethanolamine (PE) + phosphatidylserine (PS) + phosphatidylinositol (PI) (loss from phosphatidylinositol minus accumulated phosphatidate), amounted to 44.4 and 7.3 nmol/2 x 10(9) platelets (mean values, n = 4 subjects), respectively, in response to thrombin (2 units/ml). The phosphatidylcholine, phosphatidylethanolamine (including alkenylacyl), phosphatidylserine, and phosphatidylinositol contributed 46, 17, less than 5, and 33%, respectively, of the AA loss; in contrast to these distributions, the corresponding phospholipid contributions to the net loss of EPA were 71, 27, less than 1, and less than 2%, respectively. Furthermore, the inhibition of AA- and EPA-phospholipid degradation by trifluoperazine indicated that almost all of the release of EPA occurs from PC and PE (greater than 95% of total EPA loss) upon thrombin stimulation and is mediated predominantly via phospholipase A2 activity with almost no contribution from PI. Similarities in the molar ratios of AA/EPA in the PC (3.9) or PE (3.7) which were degraded with those in the corresponding phospholipids from resting platelets suggested no marked selectivity by the phospholipase A2 in intact thrombin-stimulated human platelets in the hydrolysis of AA-PC (or AA-PE) versus EPA-PC (or EPA-PE). Quantitation of the newly released free AA and EPA was determined in the presence of BW755C, a dual cyclooxygenase/lipoxygenase inhibitor which was found not to influence the degradation of individual AA- and EPA-containing phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Quantitative loss of individual eicosapentaenoyl-relative to arachidonoyl-containing phospholipids in thrombin-stimulated human platelets. 282 97

[3H]Pentoxifylline and [3H]propentofylline were taken up by human platelets in a dose-dependent manner probably involving a passive diffusion through the plasma membrane. In vitro, the two drugs were able to inhibit platelet activation induced by thrombin. serotonin secretion was reduced from 57% to 38% and 28% in the presence of 1 mM pentoxifylline and 1 mM propentofylline, respectively. Platelet aggregation was inhibited in the same way. Modifications of [14C]arachidonic acid metabolism in human platelets stimulated by thrombin were then measured in the presence of drugs. Preincubation of platelets with 1 mM pentoxifylline or propentofylline inhibited the production of [14C]arachidonic acid metabolites, without any accumulation of free arachidonic acid, suggesting an action at a step preceding its conversion. Phosphatidylinositol and phosphatidylcholine hydrolysis measured upon thrombin treatment as well as phosphatidic acid production were reduced or suppressed in the presence of the drugs. A dose-dependence study showed that phosphatidylcholine hydrolysis was totally inhibited at 5.10(-4) M propentofylline, while phosphatidic acid formation was reduced by only 40%. Propentofylline was in general more efficient than pentoxifylline in inhibiting events occurring upon thrombin stimulation. Our results suggest that the two methylxanthines inhibit both phospholipase A2 and phospholipase C, the former displaying a greater sensitivity to the two drugs.
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PMID:Effects of two methylxanthines, pentoxifylline and propentofylline, on arachidonic acid metabolism in platelets stimulated by thrombin. 284 Sep 8

R59 022 (6-[2-[4-[(4-fluorophenyl)phenylmethylene]-1- piperidinyl]ethyl]-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one) has been suggested as an inhibitor of diacylglycerol kinase in erythrocyte membranes and intact platelets. In the present study, we have investigated the effects of this drug on arachidonic acid mobilization occurring in response to thrombin in intact human platelets. Our results indicate that release of arachidonic acid from membrane phospholipids such as phosphatidylcholine and phosphatidylinositol was severely impaired by R59 022 and the extent of inhibition amounted to 77% and 84%, respectively, as compared to controls. This resulted in a dramatic decrease in the accumulation of free arachidonic acid (labeled/unlabeled) and the percent inhibition of free arachidonic acid accumulation amounted to 80-90% as compared to controls. Furthermore, the drug caused a significant accumulation of thrombin-induced diacylglycerol (labeled) without affecting the formation of labeled phosphatidic acid (PA). We found no significant changes in the radioactivity of either phosphatidylethanolamine or phosphatidylserine following stimulation with thrombin in the presence or absence of R59 022. We conclude that the observed inhibition of thrombin-induced arachidonic acid mobilization by R59 022 may be due to its effects on the activities of diacylglycerol lipase/phospholipase A2. In addition, the failure of further stimulation of thrombin-induced PA by R59 022 may indicate that PA-specific phospholipase A2 is either not involved in the release of arachidonic acid or is not a major source for arachidonic acid release in thrombin-stimulated human platelets. These findings may prove to be important when this drug is used as a selective inhibitor of diacylglycerol kinase.
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PMID:The inhibition of arachidonic acid mobilization in human platelets by R59 022, a diacylglycerol kinase inhibitor. 284 Sep 67

Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating.
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PMID:Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin. 286 Jan 11

The regulating mechanisms of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin were investigated. The formation of PAF-acether was maximal at 5 min after stimulation and gradually decreased for up to 30 min. Thrombin induced a rapid 3-4-fold increase in the activity which was maximal by 1 min after stimulation and returned progressively to basal level within 10 min. The thrombin-induced enhancement in acetyltransferase activity was due to an increase of the Vmax of the acetylation reaction without a significant effect on the apparent Km of the enzyme for acetyl-CoA. Human endothelial cells also exhibited a basal PAF-acether acetylhydrolase activity which was not altered upon thrombin stimulation. The pretreatment with 2 mM phenylmethylsulfonyl fluoride (PMSF), a serine proteinase inhibitor reported to block the acetylhydrolase, induced about 2-times more PAF-acether production in response to 2.5 U/ml thrombin stimulation. However, this enhancement of PAF-acether formation seems to be not only due to the inhibition of the acetylhydrolase, but also to the influences on the activities of the acetyltransferase and other enzymes such as phospholipase A2. These results suggest a key role for acetyltransferase and acetylhydrolase in the regulation of PAF-acether formation and catabolism in thrombin-stimulated human endothelial cells.
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PMID:Regulation of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin. 288 88

Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s).
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PMID:Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts. 290 Oct 97

Platelets, when stirred with 3 U thrombin/10(9) platelets, produced significant quantities of palmitoyllysophosphatidic acid (2.17 ng/10(9) platelets), stearoyllysophosphatidic acid (2.11 ng/10(9) platelets), and arachidonoyllysophosphatidic acid (1.06 ng/10(9) platelets). When platelets were pretreated with 100 microM of the phospholipase A2 inhibitor U10029A, there was a significant decrease in thrombin-stimulated production of stearoyllysophosphatidic acid (to 0.16 ng/10(9) platelets), while arachidonoyllysophosphatidic acid production was unchanged. U10029A concomitantly increased thrombin-stimulated production of stearoyl-containing phosphatidic acid species (primarily stearoylarachidonoylphosphatidic acid) from 5.99 to 9.71 ng/10(9) platelets. The results are consistent with the concept that stearoyllysophosphatidic acid production in platelets occurs via phospholipase A2 degradation of phosphatidic acid.
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PMID:Identification of the molecular species of lysophosphatidic acid produced when platelets are stimulated by thrombin. 291 52

Intact human platelets were stimulated with alpha or gamma thrombin in the presence and absence of epinephrine and the ability of these agonists to stimulate aggregation, arachidonic acid release and protein phosphorylation was measured. Epinephrine alone had no effect on any of these events. Both alpha and gamma thrombin induced platelet aggregation which was potentiated in each case by epinephrine. Similarly, both thrombin species were able to induce the phosphorylation of platelet 20 KDa and 47KDa proteins. The gamma thrombin-induced phosphorylation was slightly enhanced by epinephrine. In contrast, only alpha thrombin was capable of inducing significant arachidonic acid release and the small release induced by gamma thrombin was reduced by epinephrine. These results show that the agonist-induced phosphorylation of the 47KDa protein by protein kinase C does not impart the ability to activate phospholipase A2 in human platelets, and questions the suggestion that the 47KDa protein is lipocortin.
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PMID:Phosphorylation of the 47 KDa protein in gamma-thrombin-stimulated human platelets does not activate phospholipase A2: evidence against lipocortin. 294 6


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